The present invention is directed to certain substituted pyrroles that are antiproliferative agents. These compounds and their pharmaceutically acceptable salts are useful in the treatment or control of cell proliferative disorders, in particular cancer. The invention is also directed to pharmaceutical compositions containing such compounds, and to methods for the treatment and/or prevention of cancer, particularly the treatment or control of solid tumors.
Uncontrolled cell proliferation is the hallmark of cancer. Cancerous tumor cells typically have some form of damage to the genes that directly or indirectly regulate the cell-division cycle. Much research has been expended in the study of antiproliferative agents. While many agents having desired antiproliferative activities have been identified, many of these agents have various drawbacks, including poor solubility, molecular complexity, etc., which may render them either unsuitable or inconvenient for therapeutic use in human patients. There continues to be a need for small molecule compounds that may be readily synthesized, are effective as cancer therapeutic agents and are suitable for continuous infusion delivery to patients. It is thus an object of this invention to provide such compounds as well as pharmaceutical compositions containing such compounds.
As used herein, the following terms shall have the following definitions.
xe2x80x9cAlkylxe2x80x9d denotes a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 15, preferably 1 to 10, carbon atoms. Alkyl groups may be substituted as specifically provided infra. In addition the alkyl chain may include one or more hetero atoms in lieu of one or more carbon atoms. xe2x80x9cLower alkylxe2x80x9d groups having from 1 to 6, preferably 1 to 4, carbon atoms are preferred. Typical lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, 2-butyl, pentyl, hexyl, and the like.
xe2x80x9cAlkenylxe2x80x9d means a straight-chain or branched aliphatic unsaturated hydrocarbon having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, most preferably 1 to 6 carbon atoms.
xe2x80x9cAlkoxyxe2x80x9d means an alkyl group that is attached to the remainder of the molecule by oxygen (e.g. ROxe2x80x94, such as methoxy, ethoxy, etc.).
xe2x80x9cArylxe2x80x9d means an aromatic ring having 5 to 10 atoms and consisting of 1 or 2 rings, which optionally may include one or more heteroatoms that are the same or different. For the purposes of this definition, aryl includes heteroaryl. Preferred heteroatoms include nitrogen, sulfur, or oxygen, singly or in any combination, in place of one or more of the carbons. Examples of aryl groups within this definition are phenyl, pyridine, imidazole, pyrrole, triazole, furan, pyrimidine.
xe2x80x9cCycloalkylxe2x80x9d means a non-aromatic, partially or completely saturated cyclic aliphatic hydrocarbon group containing 3 to 8 atoms. Examples of cycloalkyl groups include cyclopropyl, cyclopentyl and cyclohexyl.
xe2x80x9cEffective amountxe2x80x9d means an amount of at least one compound of Formula I or a pharmaceutically acceptable salt thereof that significantly inhibits proliferation and/or prevents differentiation of a human tumor cell, including human tumor cell lines.
xe2x80x9cHetero atomxe2x80x9d means an atom selected from nitrogen, sulfur and oxygen. Hetero atoms are independently selected and may replace one or more carbon atoms.
xe2x80x9cHeterocyclexe2x80x9d means a 3- to 10-membered non-aromatic, partially or completely saturated hydrocarbon group that contains at least one hetero atom. Such ring systems include, morpholine, pyrrolidine, piperidine, piperazine
xe2x80x9cIC50xe2x80x9d refers to the concentration of a particular compound according to the invention required to inhibit 50% of a specific measured activity. IC50 can be. measured, inter alia, as is described in Example 26, infra.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d refers to conventional acid-addition salts or base-addition salts which retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and nitric acid, and those derived from organic acids such as acetic acid, tartaric acid, salicylic acid, methanesulfonic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide.
xe2x80x9cPharmaceutically acceptable,xe2x80x9d such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.
xe2x80x9cPharmaceutically active metabolitexe2x80x9d means a metabolic product of a compound of formula I which is pharmaceutically acceptable and effective.
xe2x80x9cPlasma conversionxe2x80x9d with respect to compounds of formula I means the degradation (enzymatic and/or non-enzymatic) of such compound in human or rodent plasma at 37xc2x0 C. from 30 minutes to 6 hours to give 3-(1-methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione, a pharmaceutically active metabolite of compounds of formula I, as well as pharmaceutically active metabolites thereof. This conversion is typically given as the percent degradation over a specified time frame.
xe2x80x9cPolyethylene glycolxe2x80x9d or xe2x80x9cPEGxe2x80x9d groups represent structures of the general formula HO(CH2CH2O)nR8, where n is on average between 2 and 1500, preferably 15 to 150, with an average molecular weight of 500 to 5000 Daltons, and wherein R8 is carboxy or lower alkyl, preferably methyl or ethyl. PEG groups may be reacted with compounds according to this invention to yield pegylated compounds also within the scope of this invention.
xe2x80x9cProdrugxe2x80x9d refers to a compound that may be converted under physiological conditions or by solvolysis to a pharmaceutical active compound. A prodrug may be inactive when administered to a subject but is converted in vivo to an active compound.
xe2x80x9cStabilityxe2x80x9d is an overall assessment of the ability of a compound of formula I to withstand degradation in a typical solution used for the administration of drugs intravenously. Specifically, it refers to the ability of any given compound of formula I to release 3-(1-methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione, over a 72 hour period in a mixture of acetonitrile and saline or dextrose water. The
xe2x80x9cstabilityxe2x80x9d of a compound of formula I is xe2x80x9cvery goodxe2x80x9d if the less than 1% 3-(1-Methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione is detected, xe2x80x9cgoodxe2x80x9d if less than 2.5% 3-(1-Methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione is detected, and xe2x80x9cfairxe2x80x9d if less than 5% 3-(1-Methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione is detected after 72 hours incubation at room temperature.
xe2x80x9cSubstituted,xe2x80x9d as in substituted alkyl, means that the substitution can occur at one or more positions and, unless otherwise indicated, that the substituents at each substitution site are independently selected from the specified options.
xe2x80x9cSubstituted aminoxe2x80x9d means an amino group which is mono- or di-substituted with another group, preferably lower alkyl (e.g. methyl, or ethyl).
Specifically, the invention relates to substituted pyrroles having the formula: 
and pharmaceutically acceptable salts of the foregoing compounds, wherein
R is selected from the group consisting of xe2x80x94CH2OPO3R1R2, xe2x80x94CH2OH, xe2x80x94CH2OCOR3, xe2x80x94CH2OCO2R3, xe2x80x94CH2OCONHR3, and xe2x80x94CONHR3;
R1 and R2 are selected from the group consisting of H, Na and NH4 and are the same unless either R1 or R2 is H, in which case the other can be different, or alternatively, R1 and R2 together represent calcium.
R3 is selected from the group consisting of
alkyl which optionally may be substituted by one or more substituents selected from the group consisting of xe2x80x94CO2R4, xe2x80x94NR5R6, polyethylene glycol, xe2x80x94OPO3R1R2, hydroxy, alkoxy and aryl; alkenyl which optionally may be substituted by one or more substituents selected from the group consisting of xe2x80x94CO2R4, xe2x80x94NR5R6, polyethylene glycol, xe2x80x94OPO3R1R2, hydroxy, alkoxy and aryl; cycloalkyl which optionally may be substituted by one or more substituents selected from the group consisting of xe2x80x94CO2R4, xe2x80x94NR5R6, polyethylene glycol, xe2x80x94OPO3R1R2, hydroxy, alkoxy and aryl;
heterocycle, which when including N as a heteroatom, the N optionally may be substituted with lower alkyl and xe2x80x94COR7,
aryl which optionally may be substituted by one or more substituents selected from the group consisting of CO2R4, hydroxy, alkoxy, polyethylene glycol, OPO3R1R2, and alkyl which itself may be substituted with hydroxy alkoxy, carboxy and substituted amino, provided that when aryl represents pyridine, the nitrogen may be substituted with lower alkyl;
R4 is selected from the group consisting of H, Na and lower alkyl;
R5 and R6 are each independently selected from the group consisting of H, lower alkyl, and xe2x80x94COR7, or alternatively, the group xe2x80x94NR5R6 together form a 5 or 6 membered heterocyclic ring; and
R7 is lower alkyl which optionally may be substituted with carboxy, polyethylene glycol and substituted amino.
The compounds of formula I have antiproliferative activity, specifically, they inhibit cell division in G2/M phase of the cell cycle and are generally referred to as xe2x80x9cG2/M phase cell-cyclexe2x80x9d inhibitors. These compounds are stable, soluble prodrugs of an anticancer therapeutic agent within U.S. Pat. No. 5,057,614 and are thus suitable for continuous infusion delivery.
The present invention is further directed to pharmaceutical compositions comprising a pharmaceutically effective amount of any one or more of the above-described compounds and a pharmaceutically acceptable carrier or excipient.
The present invention is also directed to a method for treating solid tumors, in particular breast or colon tumors, by administering to a human patient in need of such therapy an effective amount of a compound of formula I and/or its pharmaceutically acceptable salts.
In a preferred embodiment of the compounds of formula I, R is selected from the group consisting of xe2x80x94CH2OPO3R1R2, xe2x80x94CH2OCOR3, xe2x80x94CH2OCO2R3, xe2x80x94CH2OCONHR3, and xe2x80x94CONHR3, preferably xe2x80x94CH2OPO3R1R2, xe2x80x94CH2OCOR3 and xe2x80x94CONHR3, most preferably xe2x80x94CH2OPO3R1R2 and xe2x80x94CH2OCOR3.
In another preferred embodiment of the compounds of formula I, R is xe2x80x94CH2OCO-pyridine wherein the N atom on the pyridine is substituted with lower alkyl, most preferably methyl or ethyl, thereby creating a quaternary nitrogen atom.
In another preferred embodiment of the compounds of formula I, R1 and R2 are independently selected from the group consisting of H and Na.
In another preferred embodiment of the compounds of formula I, R3 is heterocycle containing at least one nitrogen atom that optionally may be substituted with xe2x80x94COR7.
In another preferred embodiment of the compounds of formula I, R3 is aryl which is substituted with xe2x80x94OPO3R1R2, and R1 and R2 are independently selected from H or Na.
In another preferred embodiment of the compounds of formula I, R3 is aryl which is substituted by the group consisting of xe2x80x94CO2Na, polyethylene glycol and xe2x80x94CH2CH2N(CH2CH2)2.
In another preferred embodiment of the compounds of formula I, R3 is lower alkyl which is substituted with xe2x80x94CO2Na.
In another preferred embodiment of the compounds of formula I, the group NR5R6 together forms a 5 or 6 membered heterocyclic ring, preferably piperidine or pyrrolidine.
In another preferred embodiment of the compounds of formula I, R5 and R6 are each independently selected from the group consisting of H, methyl and ethyl.
In another preferred embodiment of the compounds of formula I, R7 is ethyl which is substituted with polyethylene glycol.
In another preferred embodiment of the compounds of formula I, the polyethylene glycol has a molecular weight of from about 750 to about 5000 Daltons.
In another preferred embodiment of the compounds of formula I, R is xe2x80x94CH2OCOR3, wherein R3 is ethyl which is substituted with PEG having a molecular weight of from about 750 to about 5000 Daltons.
The following are examples of preferred compounds of formula I:
phosphoric acid mono-[3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl]sodium salt,
O-[2-[[2,5-dihydro-3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-yl]methoxycarbonyl]ethyl]-Oxe2x80x2-methylpolyethylene glycol 2000,
phosphoric acid mono-(4-{[3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrole-1-carbonyl]-amino}butyl) ester sodium salt,
1-methyl-3-[3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethoxycarbonyl]-pyridinium trifluoroacetate,
1-hydroxymethyl-3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-pyrrole-2,5-dione; and
O-[2-[[[2,5-Dihydro-3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-2,5-dioxo-1H-pyrrol-1-yl]methoxy]carbonyl]methyl]-Oxe2x80x2-methylpolyethylene glycol 2000.
The compounds disclosed herein and covered by the above formulae may exhibit tautomerism or structural isomerism. It is intended that the invention encompasses any tautomeric or structural isomeric form of these compounds, or mixtures of such forms, and is not limited to any one tautomeric or structural isomeric form utilized within the formulae drawn above.
The compounds of the invention may be prepared by processes known in the art. Suitable processes for synthesizing these compounds are provided in the examples. Generally, these compounds may be prepared according to the following synthesis schemes.
Compounds of formula I, in which R signifies CH2OCOR3, and in which R3 is as described above, may be prepared as indicated in scheme I below, provided that if R3 contains a hydroxy, hydroxyalkyl, amino, aminoalkyl, monoalkylamino, or carboxyl, such group is first protected with a conventional protective group know to those skilled in the art. Scheme I is also useful to prepare compounds of formula I wherein R is xe2x80x94CH2OH. 
As indicated in scheme Ia, a chloromethyl ester prepared by reacting a known carboxylic acid or a carboxylic acid prepared by known methods, with ClCH2OSO2Cl in methylene chloride and water, in the presence of a base such as sodium carbonate and a phase transfer catalyst such as tetrabutylammoniumhydrogen sulfate, was reacted with 3-(1-methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione [prepared as exemplified in Davis U.S. Pat. No. 5,057,614].
Alternatively, 3-(1-methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione [prepared as exemplified in Davis U.S. Pat. No. 5,057,614] is treated with formaldehyde to yield the hydroxymethyl intermediate 2. This intermediate is then esterified using known procedures. Typically, the hydroxy intermediate 2 is treated with a known carboxylic acid or a carboxylic acid prepared by known methods, in a solvent such as methylene chloride in the presence of EDC and dimethylaminopyridine for several hours at room temperature. Alternatively, the hydroxy intermediate 2 may be treated with a known acid chloride or an acid chloride prepared from known methods.
To prepare compounds of structure 3 wherein R3 contains a heteroaromatic ring, the heteroatom such as N may be further modified by reaction with an alkyl iodide such as CH3I in a solvent such as acetonitrile. Alternatively compounds of structure 3 wherein R3 contains a suitably protected hydroxy, hydroxyalkyl, amino, aminoalkyl, monoalkylamino, may be further modified by first removing the protective group by known methods. The amino or hydroxy group can then be modified to the desired amide or ester by methods known in the art.
Compounds of the general formula I in which R signifies xe2x80x94CH2OPO3R1R2, and wherein R1 and R2 are as defined above, can be prepared by the following scheme (II). 
Typically, hydroxymethyl intermediate 2 is coupled with a suitably protected phosphate by a mitsunobu reaction using triphenylphosphine and diethylazodicarboxylate to give compound of structure 4 in which Y represents a suitable protecting group. Removal of the protecting groups may be achieved by any of the standard methods to give the phosphoric acid 5. In particular, when Y represents a benzyl group, the protective groups are removed by using cyclohexadiene and palladium on carbon as a catalyst. Compound 5 can then be converted to its salt, such as a monosodium salt 6, by standard methods.
Compounds of formula I, in which R signifies xe2x80x94CH2OCO2R3, and in which R3 is as described above, were prepared according to scheme III below. 
Typically, hydroxymethyl intermediate 2 is treated with a known chloroformate or a chloroformate prepared using known procedures, in a solvent such as THF at temperatures of 50 to 20xc2x0 C., in the presence of dimethylaminopyridine and 1,5-diazabicyclo(4.3.0)non-5-ene to afford the desired carbonate.
Compounds of formula I, in which R signifies xe2x80x94CH2OCONHR3, and in which R3 is as described above, were prepared as described in scheme IV below. 
Typically, hydroxymethyl intermediate 2 is deprotonated using a strong base such as n-butyllithiuim or lithium bis(trimethylsilyl)amide in a solvent such as THF at 0xc2x0 C. The anion generated is then treated in the same solvent with bis(p-nitrophenyl)carbonate, followed by a known amine or an amine prepared using known procedures.
Compounds of formula I, in which R signifies xe2x80x94CONHR3, and in which R3 is as defined above, may be prepared as indicated in scheme V, provided that if R3 contains a hydroxy, hydroxyalkyl, amino, aminoalkyl, monoalkylamino, or carboxyl, such group is protected with a conventional protective group. 
Typically, 3-(1-Methyl-3-indolyl)-4-(1-methyl-6-nitro-3-indolyl)-1H-pyrrole-2,5-dione [prepared as exemplified in Davis U.S. Pat. No. 5,057,614] is deprotonated in an aprotic solvent such as THF at 0xc2x0 C. using a strong base such as n-butyllithiuim or lithium bis(trimethylsilyl)amide. The resulting anion is then treated with bis(p-nitrophenyl)carbonate, followed by a known amine or an amine prepared by methods known in the art.
The conversion of an acidic compound of formula I into a pharmaceutically acceptable salt can be carried out by treatment with a suitable base in a known manner. Suitable salts are those derived not only from inorganic bases, for example, sodium, potassium or calcium salts, but also from organic bases such as ethylenediamine, monoethanolamine or diethanolamine. The conversion of a basic compound of formula I into a pharmaceutically acceptable salt can be carried out by treatment with a suitable acid in a known manner. Suitable salts are those described on page 3.
In an alternative embodiment, the present invention is directed to pharmaceutical compositions comprising at least one compound of formula I or a pharmaceutically acceptable salt thereof.
These pharmaceutical compositions can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. They can also be administered rectally, for example, in the form of suppositories. In particular, however, the compounds of the present invention are suitable for parenteral administration, for example, in the form of injection solutions.
The pharmaceutical compositions of the present invention comprising compounds of formula I, prodrugs of such compounds, or the salts thereof, may be manufactured in a manner that is known in the art, e.g. by means of conventional mixing, encapsulating, dissolving, granulating, emulsifying, entrapping, dragee-making, or lyophilizing processes. These pharmaceutical preparations can be formulated with therapeutically inert, inorganic or organic carriers. Lactose, corn starch or derivatives thereof, talc, steric acid or its salts can be used as such carriers for tablets, coated tablets, dragees and hard gelatin capsules. Suitable carriers for soft gelatin capsules include vegetable oils, waxes and fats. Depending on the nature of the active substance, no carriers are generally required in the case of soft gelatin capsules. Suitable carriers for the manufacture of solutions and syrups are water, polyols, saccharose, invert sugar and glucose. Suitable carriers for injection are water, alcohols, polyols, glycerine, vegetable oils, phospholipids and surfactants. Suitable carriers for suppositories are natural or hardened oils, waxes, fats and semi-liquid polyols.
The pharmaceutical preparations can also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They can also contain other therapeutically valuable substances, including additional active ingredients other than those of formula I.
As mentioned above, the compounds of the present invention are useful in the treatment or control of cell proliferative disorders, in particular oncological disorders. These compounds and formulations containing said compounds are particularly useful in the treatment or control of solid tumors, such as, for example, breast and colon tumors.
A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and will be adjusted to the individual requirements in each particular case. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion.